PROJECT SUMMARY Medulloblastoma is the most common malignant brain tumor of childhood, and Group 3 medulloblastoma represents a molecular subtype which affects 20% of medulloblastoma patients and confers the worst prognosis. In children diagnosed with Group 3 medulloblastoma, ten-year survival is limited to 20% and metastatic dissemination is invariably fatal. Survivors of medulloblastoma experience significant neurological and cognitive complications due to the long-term toxicities of their aggressive treatment. Notch signaling is highly involved in stem cell homeostasis and has been increasingly implicated in cancer, including medulloblastoma. The current Notch pathway inhibitors in clinical trials target gamma-secretase, which cleaves the intracellular Notch protein downstream of its receptor activation. These inhibitors are unable to distinguish between the signaling pathways of different Notch family members, and result in high levels of toxicity. It is therefore imperative to delineate the functional contributions of different Notch family members to tumor growth and propagation in order to develop specific targeted therapies. Our data demonstrate that Group 3 medulloblastoma tumors possess cells that express only Notch1 or Notch2 and cells that express both. Inhibition of Notch1 signaling resulted in the loss of metastatic cancer, with the remaining primary tumor possessing less self-renewal ability, whereas inhibition of Notch2 led to increased cell apoptosis. The role of Notch2 in the growth and metastasis of Group 3 medulloblastoma stem cells remains unknown. The proposed project will determine the relationship of Notch2 to the important cancer stem cell properties of 1) self-renewal, 2) cancer metastasis formation, and 3) cell survival through the regulation of apoptosis. These distinctions are important in understanding the developmental biology of Group 3 medulloblastoma, and will improve medulloblastoma treatments by informing how targeted Notch therapies affect cancer killing.